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Glycogen synthase is an excellent in vitro substrate for protein phosphatase-1 (PP1), which is potently inhibited by the phosphorylated forms of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) and Inhibitor-1. To test the hypothesis that the activation of glycogen synthase by insulin is due to a decrease in the inhibition of PP1 by the phosphatase inhibitors, we have investigated the effects of insulin on glycogen synthesis in skeletal muscles from wild-type mice and mice lacking Inhibitor-1 and DARPP-32 as a result of targeted disruption of the genes encoding the two proteins. Insulin increased glycogen synthase activity and the synthesis of glycogen to the same extent in wild-type and knockout mice, indicating that neither Inhibitor-1 nor DARPP-32 is required for the full stimulatory effects of insulin on glycogen synthase and glycogen synthesis in skeletal muscle.

Although glucose regulates the biosynthesis of a variety of beta cell proteins at the level of translation, the mechanism responsible for this effect is unknown. We demonstrate that incubation of pancreatic islets with elevated glucose levels results in rapid and concentration-dependent phosphorylation of PHAS-I, an inhibitor of mRNA cap-binding protein, eukaryotic initiation factor (eIF)-4E. Our initial approach was to determine if this effect is mediated by the metabolism of glucose and activation of islet cell protein kinases, or whether insulin secreted from the beta cell stimulates phosphorylation of PHAS-I via an insulin-receptor mechanism as described for insulin-sensitive cells. In support of the latter mechanism, inhibitors of islet cell protein kinases A and C exert no effect on glucose-stimulated phosphorylation of PHAS-I, whereas the phosphatidylinositol 3-kinase inhibitor, wortmannin, the immunosuppressant, rapamycin, and theophylline, a phosphodiesterase inhibitor, promote marked dephosphorylation of PHAS-I. In addition, exogenous insulin and endogenous insulin secreted by the beta cell line, betaTC6-F7, increase phosphorylation of PHAS-I, suggesting that beta cells of the islet, in part, mediate this effect. Studies with beta cell lines and islets indicate that amino acids are required for glucose or exogenous insulin to stimulate the phosphorylation of PHAS-I, and amino acids alone dose-dependently stimulate the phosphorylation of PHAS-I, which is further enhanced by insulin. Furthermore, rapamycin inhibits by approximately 62% the increase in total protein synthesis stimulated by high glucose concentrations. These results indicate that glucose stimulates PHAS-I phosphorylation via insulin interacting with its own receptor on the beta cell which may serve as an important mechanism for autoregulation of protein synthesis by translation.

Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase in vitro decreased PHAS-I binding to eukaryotic initiation factor (eIF)-4E. The decrease in binding lagged behind the phosphorylation of PHAS-I in Ser64, the preferred site of MAP kinase. Binding of the Ala64 mutant of PHAS-I to eIF-4E was abolished by MAP kinase, indicating that phosphorylation of sites other than Ser64 control binding. To identify such sites, PHAS-I was phosphorylated with MAP kinase and [gamma-32P]ATP and then cleaved proteolytically before the resulting phosphopeptides were isolated by reverse phase chromatography and directly identified by amino acid sequencing. Phosphorylated residues were located by determining the cycles in which 32P was released when phosphopeptides were subjected to sequential Edman degradation. With an extended incubation in vitro, MAP kinase phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82. In rat adipocytes, the phosphorylation of all five sites was increased by insulin and decreased by rapamycin although there were differences in the magnitude of the effects. A form of PHAS-I phosphorylated exclusively in Thr36 remained bound to eIF-4E, indicating that phosphorylation of Thr36 is insufficient for dissociation of the PHAS-I.eIF-4E complex. In summary, our results indicate that multiple phosphorylation sites are involved in the control of PHAS-I. All five sites identified fit a (Ser/Thr)-Pro motif, suggesting that the phosphorylation of PHAS-I in cells is mediated by a proline-directed protein kinase.

We have investigated the effects of insulin on the phosphorylation of glycogen Phosphorylase in skeletal muscle. Rat epitrochlearis muscles were incubated in vitrowith 32Pito label cellular phosphoproteins, before being treated with hormones. Phosphorylase, Phosphorylase kinase, and glycogen synthase were immunoprecipitated under conditions that prevented changes in their phosphorylation states. Based on measurements of the activity ratio (−AMP/+AMP) and the 32P content of Phosphorylase, 4–8% of the Phosphorylase in untreated muscles appeared to be phosphor y lated. Epinephrine promoted increases of approximately 4-fold in the 32P content and activity ratio. Neither these effects nor the epinephrine-stimulated increases in phosphorylation of glycogen synthase and Phosphorylase kinase were attenuated by insulin. However, insulin at physiological concentrations rapidly decreased the 32P content of Phosphorylase in muscles incubated without epinephrine. Results from peptide mapping experiments indicate that Phosphorylase was phosphorylated at a single site in both control and hormonetreated muscles. The maximum effect of insulin on Phosphorylase represented a decrease in 32P of approximately 50%. By comparison, the 32P content of glycogen synthase and the β subunit of Phosphorylase kinase were decreased by only 20 and 16%, respectively; the 32P content of the kinase α subunit was not affected by insulin. The results provide direct evidence that insulin decreases the amount of phosphate in Phosphorylase and Phosphorylase kinase. These findings have important implications with respect to both the regulation of glycogen metabolism in skeletal muscle and the mechanism of insulin action.

Incubating 3T3-L1 adipocytes with forskolin, which increases intracellular cAMP by activating adenylate cyclase, mimicked rapamycin by attenuating the effect of insulin on stimulating the phosphorylation of four (S/T)P sites in PHAS-I, a downstream target of the mammalian target of rapamycin (mTOR) signaling pathway. To investigate the hypothesis that increasing cAMP inhibits mTOR, the protein kinase activity of mTOR was measured in an immune complex assay with recombinant PHAS-I as substrate. Both forskolin and 8-(4-chlorophenylthio)adenosine 3'-5'-monophosphate (CPT-cAMP) prevented the activation of mTOR by insulin in adipocytes, but neither agent affected mTOR activity when added directly to the immunopurified protein. In contrast, the cAMP phosphodiesterase inhibitor, theophylline, inhibited mTOR activity not only when added to intact adipocytes but also when added to immunopurified mTOR in vitro, demonstrating that certain methylxanthines are able to inhibit mTOR independently of increasing cAMP. Forskolin and CPT-cAMP blocked the effect of insulin on increasing mTOR phosphorylation, which was assessed using mTAb1, an antibody whose binding is inhibited by phosphorylation of mTOR. Although the mTAb1 epitope contains a consensus site for protein kinase B, neither agent inhibited the activation of protein kinase B produced by insulin. These findings support the interpretation that increasing cAMP attenuates the effects of insulin on PHAS-I, p70(S6K), and other downstream targets of the mTOR signaling pathway by inhibiting the phosphorylation and activation of mTOR.

Amino acids have been identified as important signaling molecules involved in pancreatic beta-cell proliferation, although the cellular mechanism responsible for this effect is not well defined. We previously reported that amino acids are required for glucose or exogenous insulin to stimulate phosphorylation of PHAS-I (phosphorylated heat- and acid-stable protein regulated by insulin), a recently discovered regulator of translation initiation during cell mitogenesis. Here we demonstrate that essential amino acids, in particular branched-chain amino acids (leucine, valine, and isoleucine), are largely responsible for mediating this effect. The transamination product of leucine, alpha-ketoisocaproic acid, also stimulates PHAS-I phosphorylation although the transamination products of isoleucine and valine are ineffective. Since amino acids are secretagogues for insulin secretion by beta-cells, we investigated whether endogenous insulin secreted by beta-cells is involved. Interestingly, branched-chain amino acids stimulate phosphorylation of PHAS-I independent of endogenous insulin secretion since genistein (10 microM) and herbimycin A (1 microM), two tyrosine kinase inhibitors in the insulin signaling pathway, exert no effect on amino acid-induced phosphorylation of PHAS-I. Furthermore, branched-chain amino acids retain their ability to induce phosphorylation of PHAS-I under conditions that block insulin secretion from beta-cells. In exploring the signaling pathway responsible for these effects, we find that rapamycin (25 nM) inhibits the ability of branched-chain amino acids to stimulate the phosphorylation of PHAS-I and p70(s6) kinase, suggesting that the mammalian target of rapamycin signaling pathway is involved. The branched-chain amino acid, leucine, also exerts similar effects on PHAS-I phosphorylation in isolated pancreatic islets. In addition, we find that amino acids are necessary for insulin-like growth factor (IGF-I) to stimulate the phosphorylation of PHAS-I indicating that a requirement for amino acids may be essential for other beta-cell growth factors in addition to insulin and IGF-I to activate this signaling pathway. We propose that amino acids, in particular branched-chain amino acids, may promote beta-cell proliferation either by stimulating phosphorylation of PHAS-I and p70(s6k) via the mammalian target of rapamycin pathway and/or by facilitating the proliferative effect mediated by growth factors such as insulin and IGF-I.

Akt is a serine/threonine kinase that requires a functional phosphatidylinositol 3-kinase to be stimulated by insulin and other growth factors. When directed to membranes by the addition of a src myristoylation sequence, Akt becomes constitutively active. In the present study, a conditionally active version of Akt was constructed by fusing the Akt containing the myristoylation sequence to the hormone binding domain of a mutant murine estrogen receptor that selectively binds 4-hydroxytamoxifen. The chimeric protein was expressed in NIH3T3 cells and was shown to be stimulated by hormone treatment 17-fold after only a 20-min treatment. This hormone treatment also stimulated an approximate 3-fold increase in the phosphorylation of the chimeric protein and a shift in its migration on SDS gels. Activation of this conditionally active Akt resulted in the rapid stimulation of the 70-kDa S6 kinase. This conditionally active Akt was also found to rapidly stimulate in these cells the phosphorylation of properties of PHAS-I, a key protein in the regulation of protein synthesis. The conditionally active Akt, when expressed in 3T3-L1 adipocytes, was also stimulated, although its rate and extent of activation was less then in the NIH3T3 cells. Its stimulation was shown to be capable of inducing glucose uptake into adipocytes by stimulating translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane.

The eukaryotic initiation factor 4E (eIF-4E)-binding proteins PHAS-I and PHAS-II were found to have overlapping but different patterns of expression in tissues. Both PHAS proteins were expressed in 3T3-L1 adipocytes, in which insulin stimulated their phosphorylation, promoted dissociation of PHAS.eIF-4E complexes, and decreased the ability of both to bind exogenous eIF-4E. The effects of insulin were attenuated by rapamycin and wortmannin, two agents that block activation of p70(S6K). Unlike PHAS-I, PHAS-II was readily phosphorylated by cAMP-dependent protein kinase in vitro; however, the effects of insulin on both PHAS proteins were attenuated by agents that increase intracellular cAMP, by cAMP derivatives, and by phosphodiesterase inhibitors. These agents also markedly inhibited the activation of p70(S6K). In summary, our results indicate that PHAS-I and -II are controlled by the mammalian target of rapamycin and p70(S6K) signaling pathway and that in 3T3-L1 adipocytes this pathway is inhibited by increased cAMP.

The eukaryotic initiation factor 4E (eIF4E)-binding protein, PHAS-I, was phosphorylated rapidly and stoichiometrically when incubated with [gamma-32P]ATP and the mammalian target of rapamycin (mTOR) that had been immunoprecipitated with an antibody, mTAb1, directed against a region near the COOH terminus of mTOR. PHAS-I was phosphorylated more slowly by mTOR obtained either by immunoprecipitation with other antibodies or by affinity purification using a rapamycin/FKBP12 resin. Adding mTAb1 to either of these preparations of mTOR increased PHAS-I phosphorylation severalfold, indicating that mTAb1 activates the mTOR protein kinase. mTAb1-activated mTOR phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82 in PHAS-I. All five of these sites fit a (Ser/Thr)-Pro motif and are dephosphorylated in response to rapamycin in rat adipocytes. Thus, our findings indicate that Pro is a determinant of the mTOR protein kinase specificity and that mTOR contributes to the phosphorylation of PHAS-I in cells.

PHAS-I is the prototype of a group of eIF4E-binding proteins that can regulate mRNA translation in response to hormones and growth factors. To investigate the importance of PHAS-I in the physiology of the intact animal, we disrupted the PHAS-I gene in mice. Tissues and cells derived from the knockout mice contained no detectable PHAS-I protein. A related protein, PHAS-II, and eIF4E were readily detectable in tissues from these animals, but neither appeared to be changed in a compensatory manner. Mice lacking PHAS-I appeared normal at birth. However, male knockout mice weighed approximately 10% less than controls at all ages, whereas female weights were similar to those of controls. Both males and females were fertile. Tissues from adult animals appeared to be normal by routine histological staining techniques, as were routine blood cell counts and chemistries. Fibroblasts derived from PHAS-I-deficient mouse embryos exhibited normal rates of growth and overall protein synthesis, responded normally to serum stimulation of ornithine decarboxylase activity and cell growth, and rapamycin inhibition of cell growth. Under these experimental conditions, PHAS-I is apparently not required for the normal development and reproductive behavior of female mice, but is required for normal body weight in male mice; the mechanisms responsible for this phenotype remain to be determined.

Rat adipocytes were incubated with [32P]phosphate to label glycogen synthase, which was rapidly immunoprecipitated from cellular extracts and cleaved using either CNBr or trypsin. All of the [32P]phosphate in synthase was recovered in two CNBr fragments, denoted CB-1 and CB-2. Isoproterenol (1 microM) rapidly decreased the synthase activity ratio (-glucose-6-P/+glucose-6-P) and stimulated the phosphorylation of both CB-1 and CB-2 by approximately 30%. Insulin opposed the decrease in activity ratio and blocked the stimulation of phosphorylation by isoproterenol. Incubating cells with insulin alone changed the 32P content of neither CB-1 nor CB-2. Trypsin fragments were separated by reverse phase liquid chromatography and divided into peak fractions, denoted F-I-F-VII in order of increasing hydrophobicity. F-V contained almost half of the [32P]phosphate and was phosphorylated when synthase was immunoprecipitated from unlabeled fat cells and incubated with [gamma-32P]ATP and the cAMP-independent protein kinase, FA/GSK-3. That F-V also had the same retention time as the skeletal muscle synthase fragment containing sites 3(a + b + c) suggests that it contains sites 3. Muscle sites 1a, 5, 1b, and 2 eluted with F-I, F-II, F-VI, and F-VII, respectively. F-V was increased approximately 25% by isoproterenol, but the largest relative increases were observed in F-I (4-fold), F-III (4-fold), and F-VI (2-fold). These results indicate that beta-adrenergic receptor activation results in increased phosphorylation of multiple sites on glycogen synthase. Insulin plus glucose decreased the overall 32P content of synthase by approximately 30%, with the largest decrease (40%) occurring in F-V. Without glucose, insulin decreased the [32P]phosphate in F-V by 17%, an effect which was balanced by increases in F-I, F-II, and F-III so that no net change in the total 32P contents of the fractions was observed. Thus, activation of glycogen synthase by the glucose transport-independent pathway seems to involve a redistribution of phosphate in the synthase subunit.

The effects of insulin and epinephrine on the phosphorylation of glycogen synthase were investigated using rat hemidiaphragms incubated with [32P]phosphate. Antibodies against rabbit skeletal muscle glycogen synthase were used for the rapid purification of the 32P-labeled enzyme under conditions that prevented changes in its state of phosphorylation. The purified material migrated as a single radioactive species (Mapp = 90,000) when subjected to electrophoresis in sodium dodecyl sulfate. Insulin decreased the [32P]phosphate content of glycogen synthase. This effect occurred rapidly (within 15 min) and was observed with physiological concentrations of insulin (25 microunits/ml). The amount of [32P]phosphate removed from glycogen synthase by either different concentrations of insulin or times of incubation with the hormone was well correlated to the extent to which the enzyme was activated. Epinephrine (10 microM) inactivated glycogen synthase and increased its content of [32P]phosphate by about 50%. Cleavage of the immunoprecipitated enzyme with cyanogen bromide yielded two major 32P-labeled fragments of apparent molecular weights equal to approximately 28,000 and 15,000. The larger fragment (Fragment II) displayed electrophoretic heterogeneity similar to that observed with the corresponding CNBr fragment (CB-2) from purified rabbit skeletal muscle glycogen synthase phosphorylated by different protein kinases. Epinephrine increased [32P]phosphate content of both fragments; however, the increase in the radioactivity of the smaller fragment (Fragment I) was more pronounced. Insulin decreased the amount of [32P] phosphate present in Fragments I and II by about 40%. The results presented provide direct evidence that both insulin and epinephrine control glycogen synthase activity by regulating the phosphate present at multiple sites on the enzyme.

Phosphorylation of rabbit skeletal muscle glycogen synthase by a cyclic nucleotide and Ca2+-independent protein kinase, PC0.7, caused the enzyme to be a better substrate for phosphorylation by another cyclic nucleotide and Ca2+-independent protein kinase, FA/GSK-3. In contrast, phosphorylation by the combination of FA/GSK-3 and cyclic AMP-dependent protein kinase led to less phosphorylation than predicted from the individual actions of the protein kinases. These results are explained in part by the existence of cooperative interactions among the phosphorylation sites of glycogen synthase. Phosphorylation by FA/GSK-3 also correlated with a reduction in the electrophoretic mobility, in the presence of sodium dodecyl sulfate, of the glycogen synthase subunit from an apparent molecular weight of 85,000-86,000 to values of 88,000 and ultimately 90,000. The synergistic phosphorylation by PC0.7 and FA/GSK-3 was associated with an increased formation of the species of reduced electrophoretic mobility. The effects on subunit mobility were also reflected in the behavior of a larger phosphorylated CNBr fragment of glycogen synthase, CB-2, which gave apparent molecular weights of 22,000-27,000 depending on its phosphorylation state.

The present experiments were performed to identify calmodulin-binding proteins phosphorylated in response to insulin. Homogenates were prepared from 32Pi-labeled rat adipocytes. After centrifugation, the supernatants (±Ca2+) were applied to calmodulin-Sepharose columns. The bound proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and phosphoproteins were visualized by autoradiography. Several proteins bound to the affinity resin in the presence of Ca2+, two bound ±Ca2+, but only one protein, Mr= 170,000 (denoted pp170), bound in the absence of Ca2+. Binding of pp170 was inhibited by adding calmodulin (micromolar) or Ca2++ (nanomolar) to extracts prior to affinity chromatography. Physiological concentrations of insulin rapidly and reversibly increased (by as much as 4-fold) 32P-labeled pp170. Phorbol 12-myristate 13-acetate (PMA) increased (up to 3-fold) phosphorylation of pp170; but 4 α-phorbol 12,13-didecanoate was without effect. Phosphorylation of pp170 in response to insulin and PMA occurred predominantly on serine residues; no phosphotyrosine was detected. Protein kinase C inhibitors attenuated PMA-stimulated phosphorylation of pp170, but had no effect on insulin-stimulated phosphorylation. Peptide mapping indicated that pp170 was phosphorylated on multiple sites and that insulin stimulated the phosphorylation of at least one site not phosphorylated in response to PMA. The results indicate that insulin and PMA stimulate the phosphorylation of pp170 via different pathways, the latter presumably via protein kinase C.

Silencing of contractile activity in muscle is known to increase the level of acetylcholine receptor on the cell surface. Both in vivo and in vitro studies indicate that modulation of receptor-specific mRNA levels plays a role in the activity-related regulation, but other mechanisms have not been explored. In this study, we examine the synthesis and post-translational fate of receptor alpha subunit in actively contracting and tetrodotoxin-inhibited rat muscle cultures. Using metabolic labeling and immunoprecipitation with subunit-specific monoclonal antibodies, we find that the increase of alpha subunit synthesis in tetrodotoxin-inactivated cultures is insufficient to account for the increased rate at which new receptors appear on the cell surface. In evaluating stages in the post-translational processing of alpha subunit, we find that in active and inactive cultures, newly synthesized subunit acquires the ability to bind alpha-bungarotoxin with the same kinetics. However, differences were noted at or preceding the stage where alpha subunit becomes assembled with the other subunits to form the 9 S receptor. In inactivated cultures, newly synthesized alpha subunit transits a 5 S precursor pool more rapidly and is assembled more efficiently than in contracting cultures. The possibility that these differences represent a type of post-translational regulation is discussed.

The motor nerves to rat soleus and epitrochlearis muscles were sectioned 1-3 days before the muscles were incubated in vitro to assess insulin action and binding. The hormonal stimulation of [U-14C]glucose into glycogen in both muscles was decreased by over 80% after 3 days of denervation. Associated with the reductions in glycogen synthesis were losses in the ability of insulin to activate glycogen synthase. Even so, denervated and control muscles bound the same amounts of 125I-labeled insulin over a wide range of insulin concentrations. Scatchard analysis indicates that denervation changed neither receptor numbers nor affinities. The results presented strongly suggest that the loss of the ability of insulin to stimulate glycogen synthesis following denervation is the result of a postreceptor defect.

We have examined phosphorylation of nerve growth factor (NGF) receptor in cultured sympathetic neurons and PC12 cells. Dissociated rat superior cervical ganglion neurons or PC12 cells were incubated with 32Pi to label cellular phosphoproteins. Membrane proteins were solubilized, and NGF receptor proteins were immunoprecipitated with the monoclonal antibody 192-IgG. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that NGF receptor components of Mr = 80,000 and Mr = 210,000 were phosphorylated. Phosphorylation of neither species was affected by treating the cells with NGF or phorbol 12-myristate 13-acetate. When the 80,000-Da protein was subjected to complete trypsin proteolysis and then analyzed by reverse phase liquid chromatography, two 32P-labeled peptides were resolved. The more hydrophobic peptide accounted for most of the 32P and contained only phosphoserine; the other peptide contained phosphoserine and phosphothreonine. No phosphotyrosine was detected in the receptor proteins. When receptor molecules from nonlabeled PC12 cells were immunoprecipitated and then incubated in vitro with [gamma-32P]ATP and the cAMP-independent protein kinase FA/GSK-3, phosphorylation occurred predominantly on serine and to a lesser extent on threonine. However, the immunoprecipitated receptor proteins neither autophosphorylated nor were they detectably phosphorylated by cAMP-dependent protein kinase, casein kinase II, or protein kinase C (the Ca2+/phospholipid-dependent enzyme). We conclude that binding units of the NGF receptor are phosphorylated constitutively in at least two sites in intact cells and that they can be phosphorylated by FA/GSK-3 in vitro.

Rat hemidiaphragms were denervated in vivo and then incubated in vitro to assess the ability of insulin to stimulate glycogen synthesis. Denervation for 1 day resulted in 50% decreases in the stimulation by insulin of [U-14C]glucose incorporation into glycogen, and in both the basal and the insulin-stimulated activity ratios (activity minus glucose-6-P/activity plus glucose-6-P) of glycogen synthase; however, the stimulation of 2-deoxyglucose uptake by insulin was not affected by 1 day of denervation. The hormonal stimulation of [U-14C]glucose into glycogen was decreased by 90% after 3 days of denervation. At this time, the stimulation of 2-deoxyglucose uptake by insulin was also reduced and the synthase activity ratios remained depressed. Consistent with its reduced effect on glucose transport, the hormone did not increase glucose-6-P in the 3-day denervated muscles. Furthermore, the Ka for activation of glycogen synthase by glucose-6-P was higher in denervated muscles, and denervation abolished the effect of insulin on decreasing the Ka. The results presented demonstrate that denervation rapidly reduces the extent to which glycogen synthase can be activated by insulin, and has a later effect on decreasing the stimulation of glucose transport. These two effects act synergistically to markedly decrease the hormonal stimulation of glycogen synthesis.

Adipocytes were incubated with [32P]phosphate to achieve steady state labeling of glycogen synthase. The enzyme was then rapidly immunoprecipitated and subjected to electrophoresis on polyacrylamide slab gels in the presence of sodium dodecyl sulfate. The 32P-labeled glycogen synthase had an apparent molecular weight ( Mapp ) equal to 90,000. All of the [32P]phosphate could be recovered in two cyanogen bromide fragments. The larger fragment, CB-2 ( Mapp = 28,000), contained about five times more [32P]phosphate than the smaller fragment, CB-1 ( Mapp = 15,500). Insulin increased the activity ratio (-glucose-6-P/+glucose-6-P) of glycogen synthase from 0.12 to 0.26, but did not decrease the amount of [32P]phosphate in the enzyme. However, insulin promoted the formation of species of CB-2 of lower Mapp , suggesting dephosphorylation of sites that affected the electrophoretic mobility of the fragment. Glucose did not affect the mobility of CB-2, but slightly increased the activity ratio and decreased the [32P] phosphate by approximately 20%. With insulin plus glucose, the increase in activity ratio was much greater than the additive effects of either agent alone. The combination decreased the [32P]phosphate in each cyanogen bromide fragment by approximately 60%, indicating that the synergistic activation was due to enhanced dephosphorylation of multiple sites. 2-Deoxyglucose also promoted dephosphorylation of glycogen synthase, decreasing the 32P content of CB-1 and CB-2 by approximately 40% each. 3-O-Methylglucose was without effect. The results presented suggest that the activation of glycogen synthase by insulin via a glucose transport-dependent pathway may involve increased intracellular glucose-6-P which promotes dephosphorylation of sites in both CB-1 and CB-2. Activation by a glucose transport-independent pathway appears to be confined to sites located in CB-2.

Incubating rat diaphragm muscles with insulin increased the glycogen synthase activity ratio (minus glucose 6-phosphate/plus glucose 6-phosphate) by approximately 2-fold. Insulin increased the activities of mitogen-activated protein (MAP) kinase and the Mr = 90,000 isoform of ribosomal protein S6 kinase (Rsk) by approximately 1.5-2.0-fold. Epidermal growth factor (EGF) was more effective than insulin in increasing MAP kinase and Rsk activity, but in contrast to insulin, EGF did not affect glycogen synthase activity. The activation of both MAP kinase and Rsk by insulin was abolished by incubating muscles with the MAP kinase kinase (MEK) inhibitor, PD 098059; however, the MEK inhibitor did not significantly reduce the effect of insulin on activating glycogen synthase. Incubating muscles with concentrations of rapamycin that inhibited activation of p70S6K abolished the activation of glycogen synthase. Insulin also increased the phosphorylation of PHAS-I (phosphorylated heat- and acid-stable protein) and promoted the dissociation of the PHAS-I*eIF-4E complex. Increasing MAP kinase activity with EGF did not mimic the effect of insulin on PHAS-I phosphorylation, and the effect of insulin on increasing MAP kinase could be abolished with the MEK inhibitor without decreasing the effect of insulin on PHAS-I. The effects of insulin on PHAS-I were attenuated by rapamycin. Thus, activation of the MAP kinase/Rsk signaling pathway appears to be neither necessary nor sufficient for insulin action on glycogen synthase and PHAS-I in rat skeletal muscle. The results indicate that the effects of insulin on increasing the synthesis of glycogen and protein in skeletal muscle, two of the most important actions of the hormone, involve a rapamycin-sensitive mechanism that may include elements of the p70S6K signaling pathway.

We have investigated the effects of insulin and motor denervation on the phosphorylation of glycogen synthase in skeletal muscle. Rat epitrochlearis muscles were denervated in vivo 3 days before the contralateral and denervated muscles were incubated in vitro with 32Pi to label sites in glycogen synthase. The 32P-labeled synthase was rapidly immunoprecipitated from extracts under conditions which prevented changes in the phosphorylation state of the enzyme. When 32P-labeled synthase from contralateral muscles was cleaved with CNBr, essentially all of the 32P was recovered in two fragments, denoted CB-1 and CB-2. Incubating these muscles with insulin decreased the 32P content of each fragment by approximately 25%, indicating that the hormone stimulated dephosphorylation of at least two sites. Peptide mapping by reverse phase high performance liquid chromatography was performed to resolve phosphorylation sites more completely. The results suggest that the enzyme was phosphorylated in sites 1a, 1b, 2, 3(a+b+c), and 5. Insulin stimulated dephosphorylation of sites in peptides presumed to contain sites 1b, 2, and 3(a+b+c). Synthase from denervated muscles appeared to contain the same amount of phosphate as enzyme from contralateral muscles, and denervation did not detectably affect the distribution of 32P within the subunit. However, denervation abolished the effect of insulin on decreasing the 32P content of synthase. The results indicate that the insulin resistance induced by denervation involves a loss in the ability of insulin to stimulate dephosphorylation of glycogen synthase.

PHAS-I levels increased 8-fold as 3T3-L1 fibroblasts differentiated into adipocytes and acquired sensitivity to insulin. Insulin increased PHAS-I protein (3.3-fold after 2 days), the rate of PHAS-I synthesis (3-fold after 1 h), and the half-life of the protein (from 1.5 to 2.5 days). Insulin also increased the phosphorylation of PHAS-I and promoted dissociation of the PHAS-I eukaryotic initiation factor-4E (eIF-4E) complex, effects that were maximal within 10 min. With recombinant [H6]PHAS-I as substrate, mitogen-activated protein (MAP) kinase was the only insulin-stimulated PHAS-I kinase detected after fractionation of extracts by Mono Q chromatography; however, MAP kinase did not readily phosphorylate [H6]PHAS-I when the [H6]PHAS-I.eIF-4E complex was the substrate. Thus, while MAP kinase may phosphorylate free PHAS-I, it is not sufficient to dissociate the complex. Moreover, rapamycin attenuated the stimulation of PHAS-I phosphorylation by insulin and markedly inhibited dissociation of PHAS-I.eIF-4E, without decreasing MAP kinase activity. Rapamycin abolished the effects of insulin on increasing phosphorylation of ribosomal protein S6 and on activating p70S6K. The MAP kinase kinase inhibitor, PD 098059, markedly decreased MAP kinase activation by insulin, but it did not change PHAS-I phosphorylation or the association of PHAS-I with eIF-4E. In summary, insulin increases the expression of PHAS-I and promotes phosphorylation of multiple sites in the protein via multiple transduction pathways, one of which is rapamycin-sensitive and independent of MAP kinase. Rapamycin may inhibit translation initiation by increasing PHAS-I binding to eIF-4E.

The control of phosphorylase levels was investigated in rat skeletal muscle cells developing in vitro. The amount of enzyme was directly measured after immunoprecipitation using specific antibodies. The rate of phosphorylase synthesis was estimated by measuring the initial rate of formation of [3H]phosphorylase after incubating cells with [3H]tyrosine. Rates of degradation were determined either from pulse-chase experiments using [3H]tyrosine or by the loss of enzymatic activity following inhibition of protein synthesis. A large increase in phosphorylase occurred at the time myoblasts were fusing into myotubes. The accumulation of enzyme was preceded by a marked increase in the synthetic rate and was associated with a severalfold increase in the half-life of the enzyme. Following fusion, the myotubes began to spontaneously contract, and shortly thereafter, decreases in both the half-life and amount of phosphorylase were observed. The paralytic agents tetrodotoxin and lidocaine were without effect on phosphorylase levels before the onset of spontaneous activity; however, both agents increased the amount of enzyme when added to contracting myotubes. Tetrodotoxin had little effect on synthesis of [3H]phosphorylase but doubled the half-life of the enzyme. These and other results indicate that the increase in phosphorylase in differentiating muscle cells results from the coordinate control of both its synthesis and degradation, and that muscle activity decreases phosphorylase by increasing its degradation.

Immunological and microanalytical methods were used to investigate the two isozymes of phosphorylase kinase, enzyme w and enzyme r, in psoas major and tibialis anterior muscles. Peptide mapping experiments indicated that the alpha subunit of enzyme w and alpha' subunit of enzyme r were structurally very similar. Both subunits were completely immunoprecipitated from muscle extracts with an antibody specific for the beta subunit of the kinase, indicating that alpha and alpha' subunits are completely assembled with beta subunits in adult muscle fibers. The relative amounts of enzymes w and r in single fibers were determined from amounts of alpha and alpha' subunits, which were detected by immunoblotting. Phosphorylase kinase and phosphorylase activities were measured in the same fibers, as well as in individual fibers from diaphragm and soleus muscles. Slow oxidative fibers were found to contain low levels of enzyme r, but almost no enzyme w. Considerably more enzyme r was present in fast oxidative-glycolytic fibers. Fast glycolytic fibers contained the most enzyme w, and the highest levels of enzyme r were found in a subgroup of such fibers. Interestingly, more than half of the fast glycolytic fibers analyzed contained both isozymes. In these fibers phosphorylase was positively correlated with enzyme w, but negatively correlated with enzyme r. Total kinase activity ranged 30-fold from the highest in one of the psoas fibers to the lowest in one of the soleus fibers and was closely correlated with the phosphorylase levels. In psoas and soleus fibers, calculated absolute maximal rates for phosphorylase b to a conversion varied almost 2,500-fold.

Mouse BC3H1 myocytes were incubated with 32Pi before acetylcholine receptors were solubilized, immunoprecipitated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 90% of the 32P found in the receptor was bound to the δ subunit. Two phosphorylation sites in this subunit were resolved by reverse phase high performance liquid chromatography after exhaustive proteolysis of the protein with trypsin. Sites 1 and 2 were phosphorylated to ∼ the same level in control cells. The divalent cation ionophore, A23187, increased 32P in site 1 by 40%, but did not affect the 32P content of site 2. In contrast, isoproterenol increased 32P in site 2 by more than 60%, while increasing 32P in site 1 by only 20%. When dephosphorylated receptor was incubated with [γ-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase, the δ subunit was phosphorylated to a maximal level of 1.6 phosphates/subunit. Approximately half of the phosphate went into site 2, with the remainder going into a site not phosphorylated in cells. The α subunit was phosphorylated more slowly, but phosphorylation of both α and δ subunits was blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. Phosphorylation of the receptor was also observed with preparations of phosphorylase kinase. In this case phosphorylation occurred in the β subunit and site 1 of the δ subunit, neither of which were phosphorylated by cAMP-dependent protein kinase. The rate of receptor phosphorylation by phosphorylase kinase was slow relative to that catalyzed by cAMP-dependent protein kinase. Therefore, it can not yet be concluded that phosphorylase kinase phosphorylates the β subunit and the δ subunit site 1 in cells. However, the results strongly support the hypothesis that phosphorylation by cAMP-dependent protein kinase accounts for phosphorylation of the α subunit and the δ subunit site 2 in response to elevations in cAMP.